Disease resistance factors

ABSTRACT

This invention relates to isolated nucleic acid fragments encoding corn ( Zea mays ), rice ( Oryza sativa ), or wheat ( Triticum aestivum ) NPR1 homologs. The invention also relates to the construction of a chimeric gene encoding all or a portion of the NPR1 homolog, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the NPR1 homolog in a transformed host cell.

[0001] This application claims the benefit of International Application No. PCT/US99/25953, filed Nov. 4, 1999, which claims priority of U.S. Provisional Application No. 60/107,242, filed Nov. 5, 1998.

FIELD OF THE INVENTION

[0002] This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding corn (Zea mays), rice (Oryza sativa) and wheat (Triticum aestivum) NPR1 polypeptides in plants and seeds.

BACKGROUND OF THE INVENTION

[0003] Pathogens annually cause billions of dollars in damage to crops worldwide. Consequently, an increasing amount of research has been dedicated to developing novel methods for controlling plant diseases. Such studies have centered on the plant's innate ability to resist pathogen invasion in an effort to support the plant's own defenses to counter pathogen attacks. One such defense mechanism under study is known as systemic acquired resistance (SAR; reviewed in Ryals et al. (1996) Plant Cell 8:1809-1819). SAR is defined as a generalized defense response, which is often induced by avirulent pathogens and provides enhanced resistance to a broad spectrum of virulent pathogens. Avirulent pathogens carry an avirulence (avr) gene whose product can be recognized by the product of a corresponding resistance (R) gene carried by plants. Such recognition triggers both a programmed cell death response, known as the hypersensitive response (HR), around the point of pathogen infection and release of a systemic SAR-inducing signal. After a rapid and localized HR, the elevated state of resistance associated with SAR is effective throughout the plant for a period of time ranging from several days to a few weeks. Coinciding with the onset of SAR is the transcriptional activation of the pathogenesis-related (PR) genes. These genes encode proteins that exhibit antimicrobial activities (Ward et al. (1991) Plant Cell 3:1085-1094).

[0004] In Arabidopsis, expression of PR-1, β-1,3-glucanase (BGL2), and PR-5 has been shown to be tightly correlated with resistance to virulent bacterial, fungal, and oomycete pathogens; therefore, these genes are used as molecular markers for SAR (Uknes et al. (1992) Plant Cell 4:645-656). The Arabidopsis NPR1 controls the onset of SAR. Mutants with defects in NPR1 fail to respond to various SAR-inducing treatments, displaying little expression of PR genes and exhibiting increased susceptibility to infections. NPR1 was cloned using a map-based approach and was found to encode a novel protein containing ankyrin repeats. The lesion in one npr1 mutant allele disrupted the ankyrin consensus sequence, suggesting that these repeats are important for NPR1 function. Transformation of the cloned wild-type NPR1 gene into npr1 mutants complemented the mutations restoring the responsiveness to SAR induction with respect to PR-gene expression and resistance to infections. This transformation also rendered the transgenic plants more resistant to infection by P. syringae in the absence of SAR induction (Cao et al. (1997) Cell 88:57-63).

[0005] Identification of cDNAs encoding NPR1 in other crops will permit its manipulation and thus the control of crop pathogens.

SUMMARY OF THE INVENTION

[0006] The present invention concerns isolated polynucleotides comprising a nucleotide sequence encoding at least a portion of an NPR1 polypeptide.

[0007] The present invention concerns isolated polynucleotides comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding an NPR1 polypeptide having at least 80% identity, based on the Clustal method of alignment, when compared to a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16. It is preferred that the identity be at least 85%, it is preferable if the identity is at least 90%, it is more preferred that the identity be at least 95%. This invention also relates to the isolated complement of such polynucleotides, wherein the complement and the polynucleotide consist of the same number of nucleotides, and the nucleotide sequences of the complement and the polynucleotide have 100% complementarity.

[0008] In a third embodiment nucleotide sequence of the isolated first polynucleotide is selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17.

[0009] In a fourth embodiment, this invention concerns an isolated polynucleotide encoding an NPR1 polypeptide.

[0010] In a fifth embodiment, this invention relates to a chimeric gene comprising the polynucleotide of the present invention.

[0011] In a sixth embodiment, the present invention concerns an isolated nucleic acid molecule that comprises at least 100 nucleotides and remains hybridized with the isolated polynucleotide of the present invention under a wash condition of 0.1×SSC, 0.1% SDS, and 65° C.

[0012] In a seventh embodiment, the invention also relates to a host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast cell or a plant cell, or prokaryotic, such as a bacterial cell. The present invention may also relate to a virus comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.

[0013] In an eighth embodiment, the invention concerns a transgenic plant comprising a polynucleotide of the present invention.

[0014] In a ninth embodiment, the invention relates to a method for transforming a cell by introducing into such cell the polynucleotide of the present invention, or a method of producing a transgenic plant by transforming a plant cell with the polynucleotide of the present invention and regenerating a plant from the transformed plant cell.

[0015] In a tenth embodiment, the invention concerns a method for producing a nucleotide fragment by selecting a nucleotide sequence comprised by a polynucleotide of the present invention and synthesizing a polynucleotide fragment containing the nucleotide sequence. It is understood that the nucleotide fragment may be produced in vitro or in vivo.

[0016] In an eleventh embodiment the invention concerns an isolated polypeptide comprising an amino acid sequence selected from the group consisting of: (a) an NPR1 polypeptide having a sequence identity of at least 80%, based on the Clustal method of alignment, when compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16. It is preferred that the identity be at least 85%, it is more preferred if the identity is at least 90%, it is preferable that the identity be at least 95%.

[0017] In a twelfth embodiment the invention relates to an isolated polypleptide selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16.

[0018] In a thirteenth embodiment, this invention concerns an isolated polypeptide having NPR1 function.

[0019] In a fourteenth embodiment, this invention relates to a method of altering the level of expression of an NPR1 in a host cell comprising: transforming a host cell with a chimeric gene of the present invention; and growing the transformed host cell under conditions that are suitable for expression of the chimeric gene.

BRIEF DESCRIPTION OF THE DRAWING AND SEQUENCE LISTINGS

[0020] The invention can be more fully understood from the following detailed description and the accompanying drawing and Sequence Listing which form a part of this application.

[0021]FIG. 1 shows a comparison of the amino acid sequences derived from corn clone cdt1c.pk001.16 (EST, SEQ ID NO: 2), corn clone p0006.cbyvc82rx (FIS, SEQ ID NO: 8), rice clone rr1.pk0001.a11 (EST, SEQ ID NO: 4; FIS, SEQ ID NO: 12; and CGS, SEQ ID NO: 16), rice clone r10n.pk0063.d10 (FIS, SEQ ID NO: 10), and wheat clone wre1n.pk0122.c2 (EST, SEQ ID NO: 6; and FIS, SEQ ID NO: 14) with the NPR1 from Arabidopsis thaliana (NCBI General Identifier No. 1773295; SEQ ID NO: 17). Amino acids conserved among all sequences are indicated by an asterisk (*) below the alignment. Dashes are used by the program to maximize the alignment. Numbers above the alignment refer to the relative amino acid position. FIG. 1A shows amino acids 1 through 120, FIG. 1B shows amino acids 121 through 240, FIG. 1C shows amino acids 241 through 360, FIG. 1D shows amino acids 361 through 480, FIG. 1E shows amino acids 481 through 600, FIG. 1F shows amino acids 601 through 659.

[0022] Table 1 lists the plant source of the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides. Table 1 also lists the source of the polypeptides to which the polypeptides of the present invention show homology to and the NCBI General Identifier Nos. of such polypeptides. Finally, Table 1 lists the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing. The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825. TABLE 1 NPR1 Homologs SEQ ID NO: Plant Clone Designation (Nucleotide) (Amino Acid) Corn cdt1c.pk001.16 1 2 Rice rr1.pk0001.a11 3 4 Wheat wre1n.pk0122.c2 5 6 Corn p0006.cbyvc82rx 7 8 Rice r10n.pk0063.d10:fis 9 10 Rice rr1.pk0001.a11:fis 11 12 Wheat wre1n.pk0122.c2:fis 13 14 Rice rr1.pk0001.a11:cgs 15 16 Arabidopsis NCBI gi 1773295 17 thaliana

[0023] The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219 (No. 2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

[0024] In the context of this disclosure, a number of terms shall be utilized. The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acid sequence”, and “nucleic acid fragment”/“isolated nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. An isolated polynucleotide of the present invention may include at least 60 contiguous nucleotides, preferably at least 40 contiguous nucleotides, most preferably at least 30 contiguous nucleotides derived from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15, or the complement of such sequences.

[0025] The term “isolated” polynucleotide refers to a polynucleotide that is substantially free from other nucleic acid sequences, such as and not limited to other chromosomal and extrachromosomal DNA and RNA. Isolated polynucleotides may be purified from a host cell in which they naturally occur. Conventional nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides. The term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.

[0026] The term “recombinant” means, for example, that a nucleic acid sequence is made by an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated nucleic acids by genetic engineering techniques.

[0027] As used herein, “substantially similar” refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide encoded by the nucleotide sequence. “Substantially similar” also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by gene silencing through for example antisense or co-suppression technology. “Substantially similar” also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties of the resulting transcript vis-a-vis the ability to mediate gene silencing or alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof. The terms “substantially similar” and “corresponding substantially” are used interchangeably herein.

[0028] Substantially similar nucleic acid fragments may be selected by screening nucleic acid fragments representing subfragments or modifications of the nucleic acid fragments of the instant invention, wherein one or more nucleotides are substituted, deleted and/or inserted, for their ability to affect the level of the polypeptide encoded by the unmodified nucleic acid fragment in a plant or plant cell. For example, a substantially similar nucleic acid fragment representing at least 30 contiguous nucleotides derived from the instant nucleic acid fragment can be constructed and introduced into a plant or plant cell. The level of the polypeptide encoded by the unmodified nucleic acid fragment present in a plant or plant cell exposed to the substantially similar nucleic fragment can then be compared to the level of the polypeptide in a plant or plant cell that is not exposed to the substantially similar nucleic acid fragment.

[0029] For example, it is well known in the art that antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by using nucleic acid fragments that do not share 100% sequence identity with the gene to be suppressed. Moreover, alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded polypeptide, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.

[0030] Consequently, an isolated polynucleotide comprising a nucleotide sequence of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 25, and the complement of such nucleotide sequences may be used in methods of selecting an isolated polynucleotide that affects the expression of an NPR1 polypeptide in a host cell. A method of selecting an isolated polynucleotide that affects the level of expression of a polypeptide in a virus or in a host cell (eukaryotic, such as plant or yeast, prokaryotic such as bacterial) may comprise the steps of: constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; measuring the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of a polypeptide or enzyme activity in a host cell that does not contain the isolated polynucleotide.

[0031] Moreover, substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

[0032] Substantially similar nucleic acid fragments of the instant invention may also be characterized by the percent identity of the amino acid sequences that they encode to the amino acid sequences disclosed herein as determined by algorithms commonly employed by those skilled in this art. Suitable nucleic acid fragments (isolated polynucleotides of the present invention) encode polypeptides that are at least about 70% identical, preferably at least about 80% identical to the amino acid sequences reported herein. Preferred nucleic acid fragments encode amino acid sequences that are at least about 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are at least about 95% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments not only have the above identities but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids. Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0033] A “substantial portion” of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification of the protein or gene that the amino acid or nucleotide sequence comprises. Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer-based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/). In general, to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene a sequence of ten or more contiguous amino acids or thirty or more contiguous nucleotides is necessary. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12 or more contiguous nucleotides may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence. The instant specification teaches amino acid and nucleotide sequences encoding polypeptides that comprise one or more particular plant proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

[0034] “Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences set forth herein. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid fragment for improved expression in a host cell, it is desirable to design the nucleic acid fragment such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

[0035] “Synthetic nucleic acid fragments” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form larger nucleic acid fragments which may then be enzymatically assembled to construct the entire desired nucleic acid fragment. “Chemically synthesized”, as related to a nucleic acid fragment, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of nucleic acid fragments may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the nucleic acid fragments can be tailored for optimal gene expression based on optimization of the nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.

[0036] “Gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign-gene” refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.

[0037] “Coding sequence” refers to a nucleotide sequence that codes for a specific amino acid sequence. “Regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0038] “Promoter” refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a nucleotide sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or may be composed of different elements derived from different promoters found in nature, or may even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg (1989) Biochemistry of Plants 15:1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity.

[0039] “Translation leader sequence” refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner and Foster (1995) Mol. Biotechnol. 3:225-236).

[0040] “3′ non-coding sequences” refer to nucleotide sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The use of different 3′ non-coding sequences is exemplified by Ingelbrecht et al. (1989) Plant Cell 1:671-680.

[0041] “RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA (mRNA)” refers to the RNA that is without introns and that can be translated into polypeptides by the cell. “cDNA” refers to DNA that is complementary to and derived from an mRNA template. The cDNA can be single-stranded or converted to double stranded form using, for example, the Klenow fragment of DNA polymerase I. “Sense-RNA” refers to an RNA transcript that includes the mRNA and so can be translated into a polypeptide by the cell. “Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (see U.S. Pat. No. 5,107,065, incorporated herein by reference). The complementarity of an antisense RNA may be with any part of the specific nucleotide sequence, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence. “Functional RNA” refers to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.

[0042] The term “operably linked” refers to the association of two or more nucleic acid fragments on a single polynucleotide so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0043] The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. “Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. “Co-suppression” refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated herein by reference).

[0044] A “protein” or “polypeptide” is a chain of amino acids arranged in a specific order determined by the coding sequence in a polynucleotide encoding the polypeptide. Each protein or polypeptide has a unique function.

[0045] “Altered levels” or “altered expression” refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.

[0046] “Mature protein” or the term “mature” when used in describing a protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed. “Precursor protein” or the term “precursor” when used in describing a protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.

[0047] A “chloroplast transit peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. “Chloroplast transit sequence” refers to a nucleotide sequence that encodes a chloroplast transit peptide. A “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys. 100:1627-1632).

[0048] “Transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol. 143:277) and particle-accelerated or “gene gun” transformation technology (Klein et al. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by reference). Thus, isolated polynucleotides of the present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell. Such a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, supp. 1987; Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Flevin et al., Plant Molecular Biology Manual, Kluwer Academic Publishers, 1990. Typically, plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.

[0049] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

[0050] “PCR” or “polymerase chain reaction” is well known by those skilled in the art as a technique used for the amplification of specific DNA segments (U.S. Pat. Nos. 4,683,195 and 4,800,159).

[0051] The present invention concerns polynucleotides comprising nucleotide sequences selected from the group consisting of: (a) first nucleotide sequence encoding an NPR1 polypeptide having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16, or (b) a second nucleotide sequence comprising the complement of the first nucleotide sequence.

[0052] Preferably, the first nucleotide sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3,5, 7, 9, 11, 13, and 15.

[0053] Nucleic acid fragments encoding at least a portion of several NPR1 polypeptides have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art. The nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).

[0054] For example, genes encoding other NPR1 s, either as cDNAs or genomic DNAs, could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired plant employing methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis). Moreover, an entire sequence can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part or all of the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.

[0055] In addition, two short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the instant sequences. Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220). Products generated by the 3′ and 5′ RACE procedures can be combined to generate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165). Consequently, a polynucleotide comprising a nucleotide sequence of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15, and the complement of such nucleotide sequences may be used in such methods to obtain a nucleic acid fragment encoding a substantial portion of an amino acid sequence of a polypeptide.

[0056] The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of an NPR1 polypeptide, preferably a substantial portion of a plant NPR1 polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 60 (preferably at least 40, most preferably at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, and 15, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of an NPR1 polypeptide.

[0057] Availability of the instant nucleotide and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen cDNA expression libraries to isolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol. 36:1-34; Maniatis).

[0058] The polynucleotides of the present invention may be used to assemble chimeric genes which may be introduced into viruses or host cells. In another embodiment, this invention concerns viruses and host cells comprising either the chimeric genes of the invention as described herein or an isolated polynucleotide of the invention as described herein. Examples of host cells which can be used to practice the invention include, but are not limited to, yeast, bacteria, and plants.

[0059] The nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed polypeptides are present at higher levels than normal or in cell types or developmental stages in which they are not normally found. This would have the effect of altering the level of pathogen resistance in those cells. The NPR1 gene in Arabidopsis thaliana is involved in acquired pathogen resistance, thus overexpression of the polynucleotides of the present invention should allow the control of crop pathogens.

[0060] Overexpression of the proteins of the instant invention may be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development. The chimeric gene may comprise promoter sequences and translation leader sequences derived from the same genes. 3′ Non-coding sequences encoding transcription termination signals may also be provided. The instant chimeric gene may also comprise one or more introns in order to facilitate gene expression.

[0061] Plasmid vectors comprising the instant isolated polynucleotide (or chimeric gene) may be constructed. The choice of plasmid vector is dependent upon the method that will be used to transform host cells. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the isolated polynucleotide or chimeric gene. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.

[0062] For some applications it may be useful to direct the instant polypeptide to different cellular compartments, or to facilitate its secretion from the cell. It is thus envisioned that the chimeric gene described above may be further supplemented by directing the coding sequence to encode the instant polypeptide with appropriate intracellular targeting sequences such as transit sequences (Keegstra (1989) Cell 56:247-253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear localization signals (Raikhel (1992) Plant Phys. 100:1627-1632) with or without removing targeting sequences that are already present. While the references cited give examples of each of these, the list is not exhaustive and more targeting signals of use may be discovered in the future.

[0063] In another embodiment, the present invention concerns a polypeptide of at least XXX amino acids that has at least XX% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16.

[0064] The instant polypeptides (or portions thereof) may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to these proteins by methods well known to those skilled in the art. The antibodies are useful for detecting the polypeptides of the instant invention in situ in cells or in vitro in cell extracts. Preferred heterologous host cells for production of the instant polypeptides are microbial hosts. Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct a chimeric gene for production of the instant polypeptides. This chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression of the encoded NPR1. An example of a vector for high level expression of the instant polypeptides in a bacterial host is provided (Example 6).

[0065] All or a substantial portion of the polynucleotides of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and used as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. For example, the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the nucleic acid fragments of the instant invention. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1:174-181) in order to construct a genetic map. In addition, the nucleic acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).

[0066] The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4:37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.

[0067] Nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).

[0068] In another embodiment, nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favor use of large clones (several to several hundred KB; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.

[0069] A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:367 1), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.

EXAMPLES

[0070] The present invention is further defined in the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0071] The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.

Example 1

[0072] Composition of cDNA Libraries; Isolation and Sequencing of cDNA Clones

[0073] cDNA libraries representing mRNAs from various corn, rice, and wheat tissues were prepared. The characteristics of the libraries are described below. TABLE 2 cDNA Libraries from Corn, Rice, and Wheat Library Tissue Clone cdt1c Corn Developing Tassel cdt1c.pk001.16 p0006 Corn Young Shoot p0006.cbyvc82rx rl0n Rice 15 Day Old Leaf* r10n.pk0063.d10 rr1 Rice Root of Two Week Old rr1.pk0001.a11 Developing Seedling wre1n Wheat Root From 7 Day Old wreln.pk0122.c2 Etiolated Seedling*

[0074] cDNA libraries may be prepared by any one of many methods available. For example, the cDNAs may be introduced into plasmid vectors by first preparing the cDNA libraries in Uni-ZAP™ XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.). The Uni-ZAP™ XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts will be contained in the plasmid vector pBluescript. In addition, the cDNAs may be introduced directly into precut Bluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into DH10B cells according to the manufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors, plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant pBluescript plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Amplified insert DNAs or plasmid DNAs are sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or “ESTs”; see Adams et al., (1991) Science 252:1651-1656). The resulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescent sequencer.

[0075] Full-insert sequence (FIS) data is generated utilizing a modified transposition protocol. Clones identified for FIS are recovered from archived glycerol stocks as single colonies, and plasmid DNAs are isolated via alkaline lysis. Isolated DNA templates are reacted with vector primed M13 forward and reverse oligonucleotides in a PCR-based sequencing reaction and loaded onto automated sequencers. Confirmation of clone identification is performed by sequence alignment to the original EST sequence from which the FIS request is made.

[0076] Confirmed templates are transposed via the Primer Island transposition kit (PE Applied Biosystems, Foster City, Calif.) which is based upon the Saccharomyces cerevisiae Ty1 transposable element (Devine and Boeke (1994) Nucleic Acids Res. 22:3765-3772). The in vitro transposition system places unique binding sites randomly throughout a population of large DNA molecules. The transposed DNA is then used to transform DH10B electro-competent cells (Gibco BRL/Life Technologies, Rockville, Md.) via electroporation. The transposable element contains an additional selectable marker (named DHFR; Fling and Richards (1983) Nucleic Acids Res. 11:5147-5158), allowing for dual selection on agar plates of only those subclones containing the integrated transposon. Multiple subclones are randomly selected from each transposition reaction, plasmid DNAs are prepared via alkaline lysis, and templates are sequenced (ABI Prism dye-terminator ReadyReaction mix) outward from the transposition event site, utilizing unique primers specific to the binding sites within the transposon.

[0077] Sequence data is collected (ABI Prism Collections) and assembled using Phred/Phrap (P. Green, University of Washington, Seattle). Phrep/Phrap is a public domain software program which re-reads the ABI sequence data, re-calls the bases, assigns quality values, and writes the base calls and quality values into editable output files. The Phrap sequence assembly program uses these quality values to increase the accuracy of the assembled sequence contigs. Assemblies are viewed by the Consed sequence editor (D. Gordon, University of Washington, Seattle).

[0078] In some of the clones the cDNA fragment corresponds to a portion of the 3′-terminus of the gene and does not cover the entire open reading frame. In order to obtain the upstream information one of two different protocols are used. The first of these methods results in the production of a fragment of DNA containing a portion of the desired gene sequence while the second method results in the production of a fragment containing the entire open reading frame. Both of these methods use two rounds of PCR amplification to obtain fragments from one or more libraries. The libraries some times are chosen based on previous knowledge that the specific gene should be found in a certain tissue and some times are randomly-chosen. Reactions to obtain the same gene may be performed on several libraries in parallel or on a pool of libraries. Library pools are normally prepared using from 3 to 5 different libraries and normalized to a uniform dilution. In the first round of amplification both methods use a vector-specific (forward) primer corresponding to a portion of the vector located at the 5′-terminus of the clone coupled with a gene-specific (reverse) primer. The first method uses a sequence that is complementary to a portion of the already known gene sequence while the second method uses a gene-specific primer complementary to a portion of the 3′-untranslated region (also referred to as UTR). In the second round of amplification a nested set of primers is used for both methods. The resulting DNA fragment is ligated into a pBluescript vector using a commercial kit and following the manufacturer's protocol. This kit is selected from many available from several vendors including Invitrogen (Carlsbad, Calif.), Promega Biotech (Madison, Wis.), and Gibco-BRL (Gaithersburg, Md.). The plasmid DNA is isolated by alkaline lysis method and submitted for sequencing and assembly using Phred/Phrap, as above.

Example 2

[0079] Identification of cDNA Clones

[0080] cDNA clones encoding NPR1 were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequences contained in the BLAST “nr” database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The cDNA sequences obtained in Example 1 were analyzed for similarity to all publicly available DNA sequences contained in the “nr” database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the “nr” database using the BLASTX algorithm (Gish and States (1993) Nat. Genet. 3:266-272) provided by the NCBI. For convenience, the P-value (probability) of observing a match of a cDNA sequence to a sequence contained in the searched databases merely by chance as calculated by BLAST are reported herein as “pLog” values, which represent the negative of the logarithm of the reported P-value. Accordingly, the greater the pLog value, the greater the likelihood that the cDNA sequence and the BLAST “hit” represent homologous proteins.

[0081] ESTs submitted for analysis are compared to the genbank database as described above. ESTs that contain sequences more 5- or 3-prime can be found by using the BLASTn algorithm (Altschul et al (1997) Nucleic Acids Res. 25:3389-3402.) against the DuPont proprietary database comparing nucleotide sequences that share common or overlapping regions of sequence homology. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences can be assembled into a single contiguous nucleotide sequence, thus extending the original fragment in either the 5 or 3 prime direction. Once the most 5-prime EST is identified, its complete sequence can be determined by Full Insert Sequencing as described in Example 1. Homologous genes belonging to different species can be found by comparing the amino acid sequence of a known gene (from either a proprietary source or a public database) against an EST database using the tBLASTn algorithm. The tBLASTn algorithm searches an amino acid query against a nucleotide database that is translated in all 6 reading frames. This search allows for differences in nucleotide codon usage between different species, and for codon degeneracy.

Example 3

[0082] Characterization of cDNA Clones Encoding NPR1

[0083] The BLASTX search using the EST sequences from clones cdt1c.pk001.16, rr1.pk0001.a11 and wre1n.pk0122.c2 revealed similarity of the proteins encoded by the cDNAs to NPR1 from Arabidopsis thaliana (NCBI General Identifier No. 1773295). The BLAST results for each of these ESTs are shown in Table 3: TABLE 3 BLAST Results for Clones Encoding Polypeptides Homologous to NPR1 BLAST pLog Score NCBI GI No. Clone Status SEQ ID NO: 1773295 cdt1c.pk001.16 EST 2 13.22 rr1.pk0001.a11 EST 4 32.30 wre1n.pk0122.c2 EST 6 15.00

[0084] The sequence of the entire cDNA insert in clones cdt1c.pk001.16, rr1.pk0001.a11, and wre1n.pk0122.c2 was obtained. Additional searching of the DuPont proprietary database allowed the identification of another corn and another rice clones encoding NPR1 and the sequence of the entire cDNA insert in these clones was also obtained. The BLAST search using the sequences from clones listed in Table 4 revealed similarity of the hypothetical proteins encoded by Arabidopsis thaliana contigs and by the cDNAs to NPR1 from Arabidopsis thaliana (NCBI General Identifier No. 1773295). Shown in Table 4 are the BLAST results for the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”), or for sequences encoding an entire protein (“CGS”): TABLE 4 BLAST Results for Sequences Encoding Polypeptides Homologous to NPR1 BLAST pLog Score Clone Status SEQ ID NO: 1773295 p0006.cbyvc82rx FIS 8 60.22 r10n.pk0063.d10:fis CGS 10 138.00 rr1.pk0001.a11:fis FIS 12 91.22 wre1n.pk0122.c2:fis FIS 14 22.52

[0085] The sequence of the entire cDNA insert in clone rr1.pk0001.a11 corresponds to a portion of the 3′-terminus of the gene and does not cover the entire open reading frame. Amplification was used to obtain nucleic acid sequence encoding the entire open reading frame of this clone. The BLASTP search using the amino acid sequences derived from the clone listed in Table 5 revealed similarity of the polypeptides encoded Arabidopsis thaliana contigs encoding hypothetical proteins and by the cDNAs to NPR1 from Arabidopsis thaliana (NCBI General Identifier No. 1773295). Shown in Table 5 are the BLAST results for the sequences encoding the entire open reading frame (“CGS”): TABLE 5 BLAST Results for Sequences Encoding Polypeptides Homologous to NPR1 BLAST pLog Score Clone Status SEQ ID NO: 1773295 rr1.pk0001.a11:cgs CGS 16 100.00

[0086]FIG. 1 presents an alignment of the amino acid sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16 and the Arabidopsis thaliana NPR1 sequence (NCBI General Identifier No. 1773295). The data in Table 6 represents a calculation of the percent identity of the amino acid sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16 and the Arabidopsis thaliana NPR1 sequence (SEQ ID NO: 17). TABLE 6 Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to NPR1 Percent Identity to Clone SEQ ID NO: 1773295 cdt1c.pk001.16 2 45.8 rr1.pk0001.a11 4 47.6 wre1n.pk0122.c2 6 40.0 p0006.cbyvc82rx 8 40.3 r10n.pk0063.d10:fis 10 42.1 rr1.pk0001.a11:fis 12 39.1 wre1n.pk0122.c2:fis 14 33.7 rr1.pk0001.a11:cgs 16 34.1

[0087] Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode two entire rice NPR1 isozymes and substantial portions of the same. The instant nucleic acid fragments also encode substantial portions of one wheat NPR1 and two wheat NPR1 isozymes. These sequences represent the first corn, rice, and wheat sequences encoding NPR1 known to Applicant.

Example 4

[0088] Expression of Chimeric Genes in Monocot Cells

[0089] A chimeric gene comprising a cDNA encoding the instant polypeptides in sense orientation with respect to the maize 27 kD zein promoter that is located 5′ to the cDNA fragment, and the 10 kD zein 3′ end that is located 3′ to the cDNA fragment, can be constructed. The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites (NcoI or SmaI) can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the digested vector pML 103 as described below. Amplification is then performed in a standard PCR. The amplified DNA is then digested with restriction enzymes NcoI and SmaI and fractionated on an agarose gel. The appropriate band can be isolated from the gel and combined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has been deposited under the terms of the Budapest Treaty at ATCC (American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209), and bears accession number ATCC 97366. The DNA segment from pML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA can be ligated at 15° C. overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E. coli XL1-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterial transformants can be screened by restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid construct would comprise a chimeric gene encoding, in the 5′ to 3′ direction, the maize 27 kD zein promoter, a cDNA fragment encoding the instant polypeptides, and the 10 kD zein 3′ region.

[0090] The chimeric gene described above can then be introduced into corn cells by the following procedure. Immature corn embryos can be dissected from developing caryopses derived from crosses of the inbred corn lines H99 and LH132. The embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1.5 mm long. The embryos are then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept in the dark at 27° C. Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryoids and embryoids borne on suspensor structures proliferates from the scutellum of these immature embryos. The embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0091] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag, Frankfurt, Germany) may be used in transformation experiments in order to provide for a selectable marker. This plasmid contains the Pat gene (see European Pat. Publication 0 242 236) which encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin. The pat gene in p35S/Ac is under the control of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812) and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0092] The particle bombardment method (Klein et al. (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells. According to this method, gold particles (1 μm in diameter) are coated with DNA using the following technique. Ten μg of plasmid DNAs are added to 50 μL of a suspension of gold particles (60 mg per mL). Calcium chloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution) are added to the particles. The suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed. The particles are resuspended in 200 μL of absolute ethanol, centrifuged again and the supernatant removed. The ethanol rinse is performed again and the particles resuspended in a final volume of 30 μL of ethanol. An aliquot (5 μL) of the DNA-coated gold particles can be placed in the center of a Kapton™ flying disc (Bio-Rad Labs). The particles are then accelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-Rad Instruments, Hercules Calif.), using a helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm.

[0093] For bombardment, the embryogenic tissue is placed on filter paper over agarose-solidified N6 medium. The tissue is arranged as a thin lawn and covered a circular area of about 5 cm in diameter. The petri dish containing the tissue can be placed in the chamber of the PDS-1000/He approximately 8 cm from the stopping screen. The air in the chamber is then evacuated to a vacuum of 28 inches of Hg. The macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1000 psi.

[0094] Seven days after bombardment the tissue can be transferred to N6 medium that contains bialophos (5 mg per liter) and lacks casein or proline. The tissue continues to grow slowly on this medium. After an additional 2 weeks the tissue can be transferred to fresh N6 medium containing bialophos. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the bialophos-supplemented medium. These calli may continue to grow when sub-cultured on the selective medium.

[0095] Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al. (1 990) Bio/Technology 8:833-839).

Example 5

[0096] Expression of Chimeric Genes in Dicot Cells

[0097] A seed-specific expression cassette composed of the promoter and transcription terminator from the gene encoding the β subunit of the seed storage protein phaseolin from the bean Phaseolus vulgaris (Doyle et al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expression of the instant polypeptides in transformed soybean. The phaseolin cassette includes about 500 nucleotides upstream (5′) from the translation initiation codon and about 1650 nucleotides downstream (3′) from the translation stop codon of phaseolin. Between the 5′ and 3′ regions are the unique restriction endonuclease sites Nco I (which includes the ATG translation initiation codon), Sma I, Kpn I and Xba I. The entire cassette is flanked by Hind III sites.

[0098] The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the expression vector. Amplification is then performed as described above, and the isolated fragment is inserted into a pUC18 vector carrying the seed expression cassette.

[0099] Soybean embryos may then be transformed with the expression vector comprising sequences encoding the instant polypeptides. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface sterilized, immature seeds of the soybean cultivar A2872, can be cultured in the light or dark at 26° C. on an appropriate agar medium for 6-10 weeks. Somatic embryos which produce secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos which multiplied as early, globular staged embryos, the suspensions are maintained as described below.

[0100] Soybean embryogenic suspension cultures can be maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.

[0101] Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS1000/HE instrument (helium retrofit) can be used for these transformations.

[0102] A selectable marker gene which can be used to facilitate soybean transformation is a chimeric gene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The seed expression cassette comprising the phaseolin 5′ region, the fragment encoding the instant polypeptides and the phaseolin 3′ region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.

[0103] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (in order): 5 μL DNA (1 μg/μL), 20 μL spermidine (0.1 M), and 50 μL CaCl₂ (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μL 70% ethanol and resuspended in 40 μL of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five μL of the DNA-coated gold particles are then loaded on each macro carrier disk.

[0104] Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60×15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.

[0105] Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.

Example 6

[0106] Expression of Chimeric Genes in Microbial Cells

[0107] The cDNAs encoding the instant polypeptides can be inserted into the T7 E. coli expression vector pBT430. This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 was constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector. Then, the Nde I site at the position of translation initiation was converted to an Nco I site using oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM in this region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

[0108] Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment may then be purified on a 1% low melting agarose gel. Buffer and agarose contain 10 μg/ml ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELase™ (Epicentre Technologies, Madison, Wis.) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 μL of water. Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs (NEB), Beverly, Mass.). The fragment containing the ligated adapters can be purified from the excess adapters using low melting agarose as described above. The vector pBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as described above. The prepared vector pBT430 and fragment can then be ligated at 16° C. for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL). Transformants can be selected on agar plates containing LB media and 100 μg/mL ampicillin. Transformants containing the gene encoding the instant polypeptides are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis.

[0109] For high level expression, a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL2 1 (DE3) (Studier et al. (1986) J. Mol. Biol. 189:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25°. Cells are then harvested by centrifugation and re-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glass beads can be added and the mixture sonicated 3 times for about 5 seconds each time with a microprobe sonicator. The mixture is centrifuged and the protein concentration of the supernatant determined. One μg of protein from the soluble fraction of the culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 17 <210> SEQ ID NO 1 <211> LENGTH: 520 <212> TYPE: DNA <213> ORGANISM: Zea mays <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (405)..(406) <221> NAME/KEY: unsure <222> LOCATION: (411) <221> NAME/KEY: unsure <222> LOCATION: (417) <221> NAME/KEY: unsure <222> LOCATION: (492) <221> NAME/KEY: unsure <222> LOCATION: (503) <221> NAME/KEY: unsure <222> LOCATION: (513) <400> SEQUENCE: 1 cgcgccgccg ccaataagtc ccccgtgtgc gccgtctccg gcggcggcgg gccgcgctcg 60 ccgttcctcc tcacccacca ctacctcccc gtcaacggcg cgtcggcgtc ggcgtcggcg 120 tcggaggccg agcgcgacca cagggtccgg cgcatgcggc gcgcgctgga cgccgccgac 180 atcgagctgg tgaagctgat ggtgatgggc gaggggctgg acctggacgc ggcgctggcc 240 gtgcactacg ccgtgcagca ctgcggccgc gacgtcgtca aggcgctgct ggagctgggc 300 gccgccgacg tcaactcccg cgccgggccc gcggggaaga cggcgctgca cctggcggcc 360 gagatggtgt cccccgacat ggtgtccgtg ctcctcgaac aacannccga ncccagngcc 420 cggacgctgg acggggtcaa cccgctcgac gttgctccgc gggctcaact cccgaagttc 480 ctcttcaagg gncgccgtgg ccngggggtc aancagaatc 520 <210> SEQ ID NO 2 <211> LENGTH: 59 <212> TYPE: PRT <213> ORGANISM: Zea mays <400> SEQUENCE: 2 Val Arg Arg Met Arg Arg Ala Leu Asp Ala Ala Asp Ile Glu Leu Val 1 5 10 15 Lys Leu Met Val Met Gly Glu Gly Leu Asp Leu Asp Ala Ala Leu Ala 20 25 30 Val His Tyr Ala Val Gln His Cys Gly Arg Asp Val Val Lys Ala Leu 35 40 45 Leu Glu Leu Gly Ala Ala Asp Val Asn Ser Arg 50 55 <210> SEQ ID NO 3 <211> LENGTH: 518 <212> TYPE: DNA <213> ORGANISM: Oryza sativa <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (424) <221> NAME/KEY: unsure <222> LOCATION: (441) <221> NAME/KEY: unsure <222> LOCATION: (488) <221> NAME/KEY: unsure <222> LOCATION: (509) <221> NAME/KEY: unsure <222> LOCATION: (517) <400> SEQUENCE: 3 gcgcgatgcc ttcctgtcgc tgctgggtta cctgtacacg ggcaagctcc ggccggcgcc 60 ggatgacgtg gtgtcctgcg ccgaccccat gtgcccgcac gactcgtgcc cgccggcgat 120 caggttcaac gtcgagcaaa tgtacgcggc gtgggcgttc aagatcaccg agctcatctc 180 gctgttccag cgacggcttc ttaacttcgt cgataagact ctagtagaag atgttcttcc 240 aattctgcaa gttgcttttc attcagagct gactccagtg cttgaaaaat gtattcggag 300 aattgcaaga tcaaatcttg ataatgtatc gttggataag gaacttcctc cagaagttgc 360 tgttcagata aaagagattc gccaaaaatc tcagccaaat gagggtgaca ccgtcatttc 420 agancctgta catgagaaaa ngggcagaag aatccacagg ggactggatt ctgatgatgt 480 tgagcttntt aagttgcttc ttaaagaant tgggatnc 518 <210> SEQ ID NO 4 <211> LENGTH: 84 <212> TYPE: PRT <213> ORGANISM: Oryza sativa <400> SEQUENCE: 4 Asp Ala Phe Leu Ser Leu Leu Gly Tyr Leu Tyr Thr Gly Lys Leu Arg 1 5 10 15 Pro Ala Pro Asp Asp Val Val Ser Cys Ala Asp Pro Met Cys Pro His 20 25 30 Asp Ser Cys Pro Pro Ala Ile Arg Phe Asn Val Glu Gln Met Tyr Ala 35 40 45 Ala Trp Ala Phe Lys Ile Thr Glu Leu Ile Ser Leu Phe Gln Arg Arg 50 55 60 Leu Leu Asn Phe Val Asp Lys Thr Leu Val Glu Asp Val Leu Pro Ile 65 70 75 80 Leu Gln Val Ala <210> SEQ ID NO 5 <211> LENGTH: 642 <212> TYPE: DNA <213> ORGANISM: Triticum aestivum <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (26) <221> NAME/KEY: unsure <222> LOCATION: (321) <221> NAME/KEY: unsure <222> LOCATION: (335) <221> NAME/KEY: unsure <222> LOCATION: (403) <221> NAME/KEY: unsure <222> LOCATION: (408) <221> NAME/KEY: unsure <222> LOCATION: (420) <221> NAME/KEY: unsure <222> LOCATION: (474) <221> NAME/KEY: unsure <222> LOCATION: (498) <221> NAME/KEY: unsure <222> LOCATION: (508) <221> NAME/KEY: unsure <222> LOCATION: (510) <221> NAME/KEY: unsure <222> LOCATION: (563) <221> NAME/KEY: unsure <222> LOCATION: (565) <221> NAME/KEY: unsure <222> LOCATION: (583) <221> NAME/KEY: unsure <222> LOCATION: (609)..(610) <221> NAME/KEY: unsure <222> LOCATION: (617) <221> NAME/KEY: unsure <222> LOCATION: (619) <400> SEQUENCE: 5 cagggccaag agtcaaataa agatangatg tgcattgaca tcctagagag ggagatgatg 60 aggaatccta tgacagcgga agattctgtc acctcacctt tattggctga tgatcttcac 120 atgaaactaa gctacctgga aaacagagtc gcgttcgcaa gactgttctt ccctgctgaa 180 gccaaggttg ccatgcaaat tgcacaagca gacgtcacac cagaagttgg tggtttttct 240 gcagcaagta cttctggtaa actgagggaa gtcgatctga atgagacgcc aagtaacaaa 300 aaacaaaagg ctgcgttcaa nggtggatgc actangcgaa aacagtggaa ctgggccgtc 360 ggtacttccc aaactgctcg caagtgctcg acaaattctt ggnagatngc ctgcctgatn 420 gccttgatcg ttcaacacaa acggcaccct gatgaacaac aggtgaagaa atcncttctc 480 aagtgaacga tgacttcnca aacatcanan agaacgggcg ataaagattt ttcggccgtc 540 taaatcctcg tctcgggata agnangggat tacagtgttc canagcaggc aaaggtcctg 600 caagccttnn ggcacancnt aacgatttca taaggggcca at 642 <210> SEQ ID NO 6 <211> LENGTH: 60 <212> TYPE: PRT <213> ORGANISM: Triticum aestivum <220> FEATURE: <221> NAME/KEY: UNSURE <222> LOCATION: (9) <400> SEQUENCE: 6 Gln Gly Gln Glu Ser Asn Lys Asp Xaa Met Cys Ile Asp Ile Leu Glu 1 5 10 15 Arg Glu Met Met Arg Asn Pro Met Thr Ala Glu Asp Ser Val Thr Ser 20 25 30 Pro Leu Leu Ala Asp Asp Leu His Met Lys Leu Ser Tyr Leu Glu Asn 35 40 45 Arg Val Ala Phe Ala Arg Leu Phe Phe Pro Ala Glu 50 55 60 <210> SEQ ID NO 7 <211> LENGTH: 1227 <212> TYPE: DNA <213> ORGANISM: Zea mays <400> SEQUENCE: 7 agttgatgag ataaaaaatt tgcgcaagaa gtcacaaact gctgatggtg atacgttcat 60 ttcggaccct gtgcatgaga aaagagtcag aagaatccac agggcacttg actctgatga 120 tgttgagctt gtgaagttgc ttcttaatga gtccgacatc acattagatg atgccaacgc 180 attacactat gctgcttctt actgtgatcc taaagttgtc tcagagctgt tagatttggc 240 aatggctaac ttaaatttga agaatagccg tgggtacaca gcactccact tggctgctat 300 gaggagagaa ccagctataa tcatgtgtct ccttaacaaa ggggcaaatg tgtcacaact 360 gacagctgat ggcaggagcg caattggtat ttgtcggagg ttaacaagag caaaagacta 420 caatacaaag atggagcagg gtcaagaatc aaataaagat aggctgtgta tagatattct 480 agagagggag atgatgcgga atcctatggc ggtggaagat gccgtcacct cgcctttgtt 540 ggcagatgat cttcacatga agcttctcta cctggaaaac agagttgcat ttgctagatt 600 gttctttcct gctgaagcca aggtcgccat gcaaatcgca caagcagaca ccacagaaga 660 attcggcggt atagttgcag ttgcagcaag cacttctggt aaactgaggg aggtggacct 720 taatgagacg ccagtgacac aaaacaaaag gctccgttca agggtagatg cactgatgaa 780 aacagtggag ctgggccgtc ggtacttccc gaactgctcg caggtgctgg acaagttcct 840 ggaggacgat ctgccggaag gtctggacca gttctacctc cagaggggca cagccgatga 900 gcagaaggtg aagaggatgc gcttctgcga gctgaaagag gacgtgctga aggcgtttag 960 caaggacaag gcggagggca gcgtgttctc gggcctgtcc tcgtcgtcgt cgtgctcgcc 1020 gccccagaag tatgcccaga ggtgatcaag gcaccagttt ttgccgtata gtttgttatc 1080 atggtcttcg agacttggac ccggacagca tatagggaca tgtacacctg tgtatgtata 1140 gtgcttacaa ttggcgtaag tagaactata tgtatggaac ataaggaaac atggcaggaa 1200 caccgtgcaa aaagatgaaa aaaaaaa 1227 <210> SEQ ID NO 8 <211> LENGTH: 325 <212> TYPE: PRT <213> ORGANISM: Zea mays <400> SEQUENCE: 8 Pro Val His Glu Lys Arg Val Arg Arg Ile His Arg Ala Leu Asp Ser 1 5 10 15 Asp Asp Val Glu Leu Val Lys Leu Leu Leu Asn Glu Ser Asp Ile Thr 20 25 30 Leu Asp Asp Ala Asn Ala Leu His Tyr Ala Ala Ser Tyr Cys Asp Pro 35 40 45 Lys Val Val Ser Glu Leu Leu Asp Leu Ala Met ala Asn Leu Asn Leu 50 55 60 Lys Asn Ser Arg Gly Tyr Thr Ala Leu His Leu Ala Ala Met Arg Arg 65 70 75 80 Glu Pro Ala Ile Ile Met Cys Leu Leu Asn Lys Gly Ala Asn Val Ser 85 90 95 Gln Leu Thr Ala Asp Gly Arg Ser Ala Ile Gly Ile Cys Arg Arg Leu 100 105 110 Thr Arg Ala Lys Asp Tyr Asn Thr Lys Met Glu Gln Gly Gln Glu Ser 115 120 125 Asn Lys Asp Arg Leu Cys Ile Asp Ile Leu Glu Arg Glu Met Met Arg 130 135 140 Asn Pro Met Ala Val Glu Asp Ala Val Thr Ser Pro Leu Leu Ala Asp 145 150 155 160 Asp Leu His Met Lys Leu Leu Tyr Leu Glu Asn Arg Val Ala Phe Ala 165 170 175 Arg Leu Phe Phe Pro Ala Glu Ala Lys Val Ala Met Gln Ile Ala Gln 180 185 190 Ala Asp Thr Thr Glu Glu Phe Gly Gly Ile Val Ala Val Ala Ala Ser 195 200 205 Thr Ser Gly Lys Leu Arg Glu Val Asp Leu Asn Glu Thr Pro Val Thr 210 215 220 Gln Asn Lys Arg Leu Arg Ser Arg Val Asp Ala Leu Met Lys Thr Val 225 230 235 240 Glu Leu Gly Arg Arg Tyr Phe Pro Asn Cys Ser Gln Val Leu Asp Lys 245 250 255 Phe Leu Glu Asp Asp Leu Pro Glu Gly Leu Asp Gln Phe Tyr Leu Gln 260 265 270 Arg Gly Thr Ala Asp Glu Gln Lys Val Lys Arg Met Arg Phe Cys Glu 275 280 285 Leu Lys Glu Asp Val Leu Lys Ala Phe Ser Lys Asp Lys Ala Glu Gly 290 295 300 Ser Val Phe Ser Gly Leu Ser Ser Ser Ser Ser Cys Ser Pro Pro Gln 305 310 315 320 Lys Tyr Ala Gln Arg 325 <210> SEQ ID NO 9 <211> LENGTH: 2194 <212> TYPE: DNA <213> ORGANISM: Oryza sativa <400> SEQUENCE: 9 cccccgggct gcaggaattc ggcacgaggc tcgggcggga ggcctcctcc tcgcctcgcc 60 tcgccacgcc gcgccgcgac gcgacgcgcc gtggtcagct ggtcgccggt gcgggtgcgg 120 gtgcgcaatg gagccgccga ccagccacgt caccaacgcg ttctccgact cggacagcgc 180 gtccgtggag gaggggggcg ccgacgcgga cgccgacgtg gaggcgctcc gccgcctctc 240 cgacaacctc gccgcggcgt tccgctcgcc cgaggacttc gcgttcctcg ccgacgcgcg 300 catcgccgtc ccgggcggcg gcggcggcgg cggcgacctg ctggtgcacc gctgcgtgct 360 ctccgcgcgg agccccttcc tgcgcggcgt cttcgcgcgc cgcgccgccg ccgccgcagg 420 cggcggcggc gaggatggcg gcgagaggct ggagctccgg gaactcctcg gcggcggcgg 480 cgaggaggtg gaggtcgggt acgaggcgct gcggctggtg ctcgactacc tctacagcgg 540 ccgcgtcggc gacctgccca aggcggcgtg cctctgcgtc gacgaggact gcgcccacgt 600 cgggtgccac cccgccgtcg cgttcatggc gcaggtcctc ttcgccgcct ccaccttcca 660 ggtcgccgag ctcaccaacc tcttccagcg gcgtctcctt gatgtccttg ataaggttga 720 ggtagataac cttctattga tcttatctgt tgccaactta tgcaacaaat cttgcatgaa 780 actgcttgaa agatgccttg atatggtagt ccggtcaaac cttgacatga ttactcttga 840 gaagtcattg cctccagatg ttatcaagca gattattgat gcacgcctaa gcctcggatt 900 aatttcacca gaaaacaagg gatttcctaa caaacatgtg aggaggatac acagagccct 960 tgactctgac gatgtagagc tagtcaggat gctgctcact gaaggacaga caaatcttga 1020 tgatgcgttt gcactgcact acgccgtcga acattgtgac tccaaaatta caaccgagct 1080 tttggatctc gcacttgcag atgttaatca tagaaaccca agaggttata ctgttcttca 1140 cattgctgcg aggcgaagag agcctaaaat cattgtctcc cttttaacca agggggctcg 1200 gccagcagat gttacattcg atgggagaaa agcggttcaa atctcaaaaa gactaacaaa 1260 acaaggggat tactttgggg ttaccgaaga aggaaaacct tctccaaaag ataggttatg 1320 tattgaaata ctggagcaag ctgaaagaag ggacccacaa ctcggagaag catcagtttc 1380 tcttgcaatg gcaggtgaga gtctacgagg aaggttgctg tatcttgaaa accgagttgc 1440 tttggcgagg attatgtttc cgatggaggc aagagtagca atggatattg ctcaagtgga 1500 tggaactttg gaatttaacc tgggttctgg tgcaaatcca cctcctgaaa gacaacggac 1560 aactgttgat ctaaatgaaa gtcctttcat aatgaaagaa gaacacttag ctcggatgac 1620 ggcactctcc aaaacagtgg agctcgggaa acgctttttc ccgcgatgtt cgaacgtgct 1680 cgacaagatc atggatgatg aaactgatcc ggtttccctc ggaagagaca cgtccgcgga 1740 gaagaggaag aggtttcatg acctgcagga tgttcttcag aaggcattcc acgaggacaa 1800 ggaggagaat gacaggtcgg ggctctcgtc gtcgtcgtca tcgacatcga tcggggccat 1860 tcgaccaagg agatgaacac cattgctccc aaatagttgc catattgata gctaactgtc 1920 ctcctggagc tactcacctg atggttgcct tctgtcaatt gccccccaaa tatattctca 1980 atggtttagg cttgtacagt attagttctt acagctattg ccccgtcaat tgtgaaacgc 2040 agaagtttca ctagtgcttg tactcgaggt gtaatacaag tgcttgaatt ttgagttgta 2100 cttggaattt ccagtggttt gctcgtaaaa atgagatgat ttcttggctc ccaaaaaaaa 2160 aaaaaaaaaa aactcgaggg ggggcccggt accc 2194 <210> SEQ ID NO 10 <211> LENGTH: 582 <212> TYPE: PRT <213> ORGANISM: Oryza sativa <400> SEQUENCE: 10 Met Glu Pro Pro Thr Ser His Val Thr Asn Ala Phe Ser Asp Ser Asp 1 5 10 15 Ser Ala Ser Val Glu Glu Gly Gly Ala Asp Ala Asp Ala Asp Val Glu 20 25 30 Ala Leu Arg Arg Leu Ser Asp Asn Leu Ala Ala Ala Phe Arg Ser Pro 35 40 45 Glu Asp Phe Ala Phe Leu Ala Asp Ala Arg Ile Ala Val Pro Gly Gly 50 55 60 Gly Gly Gly Gly Gly Asp Leu Leu Val His Arg Cys Val Leu Ser Ala 65 70 75 80 Arg Ser Pro Phe Leu Arg Gly Val Phe Ala Arg Arg Ala Ala Ala Ala 85 90 95 Ala Gly Gly Gly Gly Glu Asp Gly Gly Glu Arg Leu Glu Leu Arg Glu 100 105 110 Leu Leu Gly Gly Gly Gly Glu Glu Val Glu Val Gly Tyr Glu Ala Leu 115 120 125 Arg Leu Val Leu Asp Tyr Leu Tyr Ser Gly Arg Val Gly Asp Leu Pro 130 135 140 Lys Ala Ala Cys Leu Cys Val Asp Glu Asp Cys Ala His Val Gly Cys 145 150 155 160 His Pro Ala Val Ala Phe Met Ala Gln Val Leu Phe Ala Ala Ser Thr 165 170 175 Phe Gln Val Ala Glu Leu Thr Asn Leu Phe Gln Arg Arg Leu Leu Asp 180 185 190 Val Leu Asp Lys Val Glu Val Asp Asn Leu Leu Leu Ile Leu Ser Val 195 200 205 Ala Asn Leu Cys Asn Lys Ser Cys Met Lys Leu Leu Glu Arg Cys Leu 210 215 220 Asp Met Val Val Arg Ser Asn Leu Asp Met Ile Thr Leu Glu Lys Ser 225 230 235 240 Leu Pro Pro Asp Val Ile Lys Gln Ile Ile Asp Ala Arg Leu Ser Leu 245 250 255 Gly Leu Ile Ser Pro Glu Asn Lys Gly Phe Pro Asn Lys His Val Arg 260 265 270 Arg Ile His Arg Ala Leu Asp Ser Asp Asp Val Glu Leu Val Arg Met 275 280 285 Leu Leu Thr Glu Gly Gln Thr Asn Leu Asp Asp Ala Phe Ala Leu His 290 295 300 Tyr Ala Val Glu His Cys Asp Ser Lys Ile Thr Thr Glu Leu Leu Asp 305 310 315 320 Leu Ala Leu Ala Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val 325 330 335 Leu His Ile Ala Ala Arg Arg Arg Glu Pro Lys Ile Ile Val Ser Leu 340 345 350 Leu Thr Lys Gly Ala Arg Pro Ala Asp Val Thr Phe Asp Gly Arg Lys 355 360 365 Ala Val Gln Ile Ser Lys Arg Leu Thr Lys Gln Gly Asp Tyr Phe Gly 370 375 380 Val Thr Glu Glu Gly Lys Pro Ser Pro Lys Asp Arg Leu Cys Ile Glu 385 390 395 400 Ile Leu Glu Gln Ala Glu Arg Arg Asp Pro Gln Leu Gly Glu Ala Ser 405 410 415 Val Ser Leu Ala Met Ala Gly Glu Ser Leu Arg Gly Arg Leu Leu Tyr 420 425 430 Leu Glu Asn Arg Val Ala Leu Ala Arg Ile Met Phe Pro Met Glu Ala 435 440 445 Arg Val Ala Met Asp Ile Ala Gln Val Asp Gly Thr Leu Glu Phe Asn 450 455 460 Leu Gly Ser Gly Ala Asn Pro Pro Pro Glu Arg Gln Arg Thr Thr Val 465 470 475 480 Asp Leu Asn Glu Ser Pro Phe Ile Met Lys Glu Glu His Leu Ala Arg 485 490 495 Met Thr Ala Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro 500 505 510 Arg Cys Ser Asn Val Leu Asp Lys Ile Met Asp Asp Glu Thr Asp Pro 515 520 525 Val Ser Leu Gly Arg Asp Thr Ser Ala Glu Lys Arg Lys Arg Phe His 530 535 540 Asp Leu Gln Asp Val Leu Gln Lys Ala Phe His Glu Asp Lys Glu Glu 545 550 555 560 Asn Asp Arg Ser Gly Leu Ser Ser Ser Ser Ser Ser Thr Ser Ile Gly 565 570 575 Ala Ile Arg Pro Arg Arg 580 <210> SEQ ID NO 11 <211> LENGTH: 2069 <212> TYPE: DNA <213> ORGANISM: Oryza sativa <220> FEATURE: <221> NAME/KEY: unsure <222> LOCATION: (65) <400> SEQUENCE: 11 gttgtrtgga attgtgagcg ataacaattt macacaggaa acagctatga ccatgattac 60 gccangcgmg caattaaccs tcactaaagg gaacaaaagc tggagcwcca ccgcggtggc 120 ggccgctcta gaavtagtgg atcccccggg ctgcaggaat tcggcacgag gcgcgatgcc 180 ttcctgtcgc tgctgggtta cctgtacacg ggcaagctcc ggccggcgcc ggatgacgtg 240 gtgtcctgcg ccgaccccat gtgcccgcac gactcgtgcc cgccggcgat caggttcaac 300 gtcgagcaaa tgtacgcggc gtgggcgttc aagatcaccg agctcatctc gctgttccag 360 cgacggcttc ttaacttcgt cgataagact ctagtagaag atgttcttcc aattctgcaa 420 gttgcttttc attcagagct gactccagtg cttgaaaaat gtattcggag aattgcaaga 480 tcaaatcttg ataatgtatc gttggataag gaacttcctc cagaagttgc tgttcagata 540 aaagagattc gccaaaaatc tcagccaaat gagggtgaca ccgtcatttc agaccctgta 600 catgagaaaa gggtcagaag aatccacagg gcactggatt ctgatgatgt tgagcttgtg 660 aagttgcttc ttaacgaatc tgagatcacc ttggatgatg ccaatgcatt gcactatgct 720 gctgcttact gtgattcgaa agttgtttcg gagttgttag acttgagact tgccaacttg 780 aatttgaaga attcgcgtgg atacacggca ctccatctgg ctgctatgag gagagagcca 840 gctattatca tgtgtctcct aaacaaagga gcagctgtat cacaattgac tgctgatggc 900 cagagtgcaa tgagtatctg ccggaggtta acaaggatga aagactacaa tacaaagatg 960 gagcaaggcc aagagtcaaa caaagacaga ttatgtattg atatattaga tagggagatg 1020 ataaggaaac ctatggcagt ggaagattct gtcacctcgc ctttgttggc tgacgatctt 1080 cacatgaagc ttctctacct tgaaaacaga gttgcatttg caagattatt ttttcctgca 1140 gaagcaaagg ttgcaatgca aattgcacaa gcagacacca caccagaatt tggcattgtt 1200 cctgcagcta gcacttctgg aaaattgaag gaagtcgatc tgaacgagac accagtaaca 1260 caaaacaaaa ggctccgttc aagggtggat gcactcatga aaacagttga gctgggacgt 1320 cgctacttcc ctaactgctc gcaggtgctc gacaaatttc tggaggatga tttgcccgat 1380 agtcctgatg cactcgacct ccaaaatggc acttctgatg agcaaaatgt taaaaggatg 1440 cggttctgtg agttaaagga ggatgtgcgc aaggcattca gcaaagacag agctgataat 1500 agcatgtttt ctatcttgtc atcttcatcg tcatcttcgc cacctcccaa ggttgcaaag 1560 aaatgacaga agttttgtaa caaatttccg ctcgtgatgt tactgggaca agagatatcg 1620 atcaatagac ctgtatagtc ttacagtggt ataacaatta gatatcgaag cttcttcgaa 1680 tattagaaag tgctgttctg ggctgcactc agctggttta tgggacccat gcggtgaaac 1740 tggcaaaaga aaaccagctg attagaggct ccaaagcagt gtctctcgtg aatatgtttg 1800 tagcattctg ttttgttcag gatggctata atgataaaat cttttcaata gatatatagc 1860 taattgtctc gtaaaaaaaa awaaaaaaaa aaaagggggg gcccggtacc caattcgccc 1920 tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa 1980 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt 2040 aatagcgaag aggcccgcac cgatcgccc 2069 <210> SEQ ID NO 12 <211> LENGTH: 455 <212> TYPE: PRT <213> ORGANISM: Oryza sativa <400> SEQUENCE: 12 Asp Ala Phe Leu Ser Leu Leu Gly Tyr Leu Tyr Thr Gly Lys Leu Arg 1 5 10 15 Pro Ala Pro Asp Asp Val Val Ser Cys Ala Asp Pro Met Cys Pro His 20 25 30 Asp Ser Cys Pro Pro Ala Ile Arg Phe Asn Val Glu Gln Met Tyr Ala 35 40 45 Ala Trp Ala Phe Lys Ile Thr Glu Leu Ile Ser Leu Phe Gln Arg Arg 50 55 60 Leu Leu Asn Phe Val Asp Lys Thr Leu Val Glu Asp Val Leu Pro Ile 65 70 75 80 Leu Gln Val Ala Phe His Ser Glu Leu Thr Pro Val Leu Glu Lys Cys 85 90 95 Ile Arg Arg Ile Ala Arg Ser Asn Leu Asp Asn Val Ser Leu Asp Lys 100 105 110 Glu Leu Pro Pro Glu Val Ala Val Gln Ile Lys Glu Ile Arg Gln Lys 115 120 125 Ser Gln Pro Asn Glu Gly Asp Thr Val Ile Ser Asp Pro Val His Glu 130 135 140 Lys Arg Val Arg Arg Ile His Arg Ala Leu Asp Ser Asp Asp Val Glu 145 150 155 160 Leu Val Lys Leu Leu Leu Asn Glu Ser Glu Ile Thr Leu Asp Asp Ala 165 170 175 Asn Ala Leu His Tyr Ala Ala Ala Tyr Cys Asp Ser Lys Val Val Ser 180 185 190 Glu Leu Leu Asp Leu Arg Leu Ala Asn Leu Asn Leu Lys Asn Ser Arg 195 200 205 Gly Tyr Thr Ala Leu His Leu Ala Ala Met Arg Arg Glu Pro Ala Ile 210 215 220 Ile Met Cys Leu Leu Asn Lys Gly Ala Ala Val Ser Gln Leu Thr Ala 225 230 235 240 Asp Gly Gln Ser Ala Met Ser Ile Cys Arg Arg Leu Thr Arg Met Lys 245 250 255 Asp Tyr Asn Thr Lys Met Glu Gln Gly Gln Glu Ser Asn Lys Asp Arg 260 265 270 Leu Cys Ile Asp Ile Leu Asp Arg Glu Met Ile Arg Lys Pro Met Ala 275 280 285 Val Glu Asp Ser Val Thr Ser Pro Leu Leu Ala Asp Asp Leu His Met 290 295 300 Lys Leu Leu Tyr Leu Glu Asn Arg Val Ala Phe Ala Arg Leu Phe Phe 305 310 315 320 Pro Ala Glu Ala Lys Val Ala Met Gln Ile Ala Gln Ala Asp Thr Thr 325 330 335 Pro Glu Phe Gly Ile Val Pro Ala Ala Ser Thr Ser Gly Lys Leu Lys 340 345 350 Glu Val Asp Leu Asn Glu Thr Pro Val Thr Gln Asn Lys Arg Leu Arg 355 360 365 Ser Arg Val Asp Ala Leu Met Lys Thr Val Glu Leu Gly Arg Arg Tyr 370 375 380 Phe Pro Asn Cys Ser Gln Val Leu Asp Lys Phe Leu Glu Asp Asp Leu 385 390 395 400 Pro Asp Ser Pro Asp Ala Leu Asp Leu Gln Asn Gly Thr Ser Asp Glu 405 410 415 Gln Asn Val Lys Arg Met Arg Phe Cys Glu Leu Lys Glu Asp Val Arg 420 425 430 Lys Ala Phe Ser Lys Asp Arg Ala Asp Asn Ser Met Phe Ser Ile Leu 435 440 445 Ser Ser Ser Ser Ser Ser Ser 450 455 <210> SEQ ID NO 13 <211> LENGTH: 1052 <212> TYPE: DNA <213> ORGANISM: Triticum aestivum <400> SEQUENCE: 13 gcacgagcag ggccaagagt caaataaaga taggatgtgc attgacatcc tagagaggga 60 gatgatgagg aatcctatga cagcggaaga ttctgtcacc tcacctttat tggctgatga 120 tcttcacatg aaactaagct acctggaaaa cagagtcgcg ttcgcaagac tgttcttccc 180 tgctgaagcc aaggttgcca tgcaaattgc acaagcagac gtcacaccag aagttggtgg 240 tttttctgca gcaagtactt ctggtaaact gagggaagtc gatctgaatg agacgccagt 300 aacaaaaaac aaaaggctgc gttcaagggt ggatgcacta gcgaaaacag tggaactggg 360 ccgtcggtac ttcccaaact gctcgcaggt gctcgacaaa ttcttggaag atggcctgcc 420 tgatggcctt gatgcgttcc agcagcaaag cggcacccct gatgagcaac aggtgaagaa 480 gatgcgcttc tgcgaggtga aggaggacgt gcgcaaagca tacagcaaag acacggccga 540 taacagcatg ttttcggccc tgtcgtcaaa ctcctcgtcc tcggcgatga agtgaaggta 600 ctgtaacagg ctgttttctc gagatgtcag ggctaaagag ggatcgctgg tcatgcgcat 660 gtatagtgcc caccatcgtg taaaaccgaa tatgaacatg aaaggaggcc ccaaaatagt 720 agaagatgat atatactttg ctggacttgg agtttgttgg agaaggctgt gccatcccat 780 cccagattcc caatatcaat ttcccatgct ggttgcgaag acggagccgt ggatcatcca 840 gcttcgacgc tatgcatgcg tgcagcctgc tgtgtttgtt tcgcatagct gcaatactta 900 tatgtttaat aatactagag agtagtaggc aattgaggct gtagcggaag ttggaaccta 960 ccttaatgta agtgaaaggg gacagttgcc ctttgtcgaa ctgttgttat caatacatag 1020 ttgattttcg taaaaaaaaa aaaaaaaaaa aa 1052 <210> SEQ ID NO 14 <211> LENGTH: 193 <212> TYPE: PRT <213> ORGANISM: Triticum aestivum <400> SEQUENCE: 14 Glu Gln Gly Gln Glu Ser Asn Lys Asp Arg Met Cys Ile Asp Ile Leu 1 5 10 15 Glu Arg Glu Met Met Arg Asn Pro Met Thr Ala Glu Asp Ser Val Thr 20 25 30 Ser Pro Leu Leu Ala Asp Asp Leu His Met Lys Leu Ser Tyr Leu Glu 35 40 45 Asn Arg Val Ala Phe Ala Arg Leu Phe Phe Pro Ala Glu Ala Lys Val 50 55 60 Ala Met Gln Ile Ala Gln Ala Asp Val Thr Pro Glu Val Gly Gly Phe 65 70 75 80 Ser Ala Ala Ser Thr Ser Gly Lys Leu Arg Glu Val Asp Leu Asn Glu 85 90 95 Thr Pro Val Thr Lys Asn Lys Arg Leu Arg Ser Arg Val Asp Ala Leu 100 105 110 Ala Lys Thr Val Glu Leu Gly Arg Arg Tyr Phe Pro Asn Cys Ser Gln 115 120 125 Val Leu Asp Lys Phe Leu Glu Asp Gly Leu Pro Asp Gly Leu Asp Ala 130 135 140 Phe Gln Gln Gln Ser Gly Thr Pro Asp Glu Gln Gln Val Lys Lys Met 145 150 155 160 Arg Phe Cys Glu Val Lys Glu Asp Val Arg Lys Ala Tyr Ser Lys Asp 165 170 175 Thr Ala Asp Asn Ser Met Phe Ser Ala Leu Ser Ser Asn Ser Ser Ser 180 185 190 Ser <210> SEQ ID NO 15 <211> LENGTH: 2717 <212> TYPE: DNA <213> ORGANISM: Oryza sativa <400> SEQUENCE: 15 ggatcccccg ggctgcagga attcggcacg agctcgtcgt cttcctccca tttttttttc 60 ctcctcctcc tcctctcatc cctcgccgcg agccaaagcc cctggtttcc tcgcaactgc 120 ctccccgcga ttccgtttga cccccactgt tcttctcccc taccaccacc aggtcgcagt 180 cgcttccaat ttccaaataa ttccctccac tccggccgct cgcgaggaaa gaaaaggatt 240 tctttttctc tctctctctc tctccccctc tctccgagat ccgtttccca aacaggcggg 300 gggtcgaaag tgtttggtac tttggtttgg ggagcttgtt tgccgacgcg gatctgcgtg 360 gagacgagca gaggggggag cgccggaatt gggtggtttg gcccgggagg cgccggaaag 420 tgggggagcc tttggattcc ccgaacccgc catggtgatc cggcacgagt agtagtggtg 480 gtggtggtat tagtagcagt gagatgccgg cgcgtagcgc ggtggtggta atagccatgg 540 agccctcgtc gtccatcacc atcgcgtcgt cgtcctcgta cctctcgaac gggtctagcc 600 cgtgctcggt ctctcttgcg ccgccggggg caggggcggt ggcggcgcag gcggcgccgg 660 ttgccgccgg ggagggcggc ggcggcggag gaggaggagg aggaggaggg agtagtagcg 720 tggaggtggt gagcctgaat cggcttagcg ctaacctcga gcggctcctc ctcgattccg 780 acctcgactg cagcgacgcc gacgtcgacg tggccgacgg tggcccgccc gtgccagtcc 840 accgctgcat cctcgccgcc cgcagcacct tcttctacaa cctcttcgcg gcgcgcggcc 900 gcggcggcga tggggctgcc ggcggcggcg gcggcggcgg tggtggggga ggggagagga 960 ctggggggag gccgcggtac aagatggagg agctcgtgcc gggaggccgc gtggggcgcg 1020 atgccttcct gtcgctgctg ggttacctgt acacgggcaa gctccggccg gcgccggatg 1080 acgtggtgtc ctgcgccgac cccatgtgcc cgcacgactc gtgcccgccg gcgatcaggt 1140 tcaacgtcga gcaaatgtac gcggcgtggg cgttcaagat caccgagctc atctcgctgt 1200 tccagcgacg gcttcttaac ttcgtcgata agactctagt agaagatgtt cttccaattc 1260 tgcaagttgc ttttcattca gagctgactc cagtgcttga aaaatgtatt cggagaattg 1320 caagatcaaa tcttgataat gtatcgttgg ataaggaact tcctccagaa gttgctgttc 1380 agataaaaga gattcgccaa aaatctcagc caaatgaggg tgacaccgtc atttcagacc 1440 ctgtacatga gaaaagggtc agaagaatcc acagggcact ggattctgat gatgttgagc 1500 ttgtgaagtt gcttcttaac gaatctgaga tcaccttgga tgatgccaat gcattgcact 1560 atgctgctgc ttactgtgat tcgaaagttg tttcggagtt gttagacttg agacttgcca 1620 acttgaattt gaagaattcg cgtggataca cggcactcca tctggctgct atgaggagag 1680 agccagctat tatcatgtgt ctcctaaaca aaggagcagc tgtatcacaa ttgactgctg 1740 atggccagag tgcaatgagt atctgccgga ggttaacaag gatgaaagac tacaatacaa 1800 agatggagca aggccaagag tcaaacaaag acagattatg tattgatata ttagataggg 1860 agatgataag gaaacctatg gcagtggaag attctgtcac ctcgcctttg ttggctgacg 1920 atcttcacat gaagcttctc taccttgaaa acagagttgc atttgcaaga ttattttttc 1980 ctgcagaagc aaaggttgca atgcaaattg cacaagcaga caccacacca gaatttggca 2040 ttgttcctgc agctagcact tctggaaaat tgaaggaagt cgatctgaac gagacaccag 2100 taacacaaaa caaaaggctc cgttcaaggg tggatgcact catgaaaaca gttgagctgg 2160 gacgtcgcta cttccctaac tgctcgcagg tgctcgacaa atttctggag gatgatttgc 2220 ccgatagtcc tgatgcactc gacctccaaa atggcacttc tgatgagcaa aatgttaaaa 2280 ggatgcggtt ctgtgagtta aaggaggatg tgcgcaaggc attcagcaaa gacagagctg 2340 ataatagcat gttttctatc ttgtcatctt catcgtcatc ttcgccacct cccaaggttg 2400 caaagaaatg acagaagttt tgtaacaaat ttccgctcgt gatgttactg ggacaagaga 2460 tatcgatcaa tagacctgta tagtcttaca gtggtataac aattagatat cgaagcttct 2520 tcgaatatta gaaagtgctg ttctgggctg cactcagctg gtttatggga cccatgcggt 2580 gaaactggca aaagaaaacc agctgattag aggctccaaa gcagtgtctc tcgtgaatat 2640 gtttgtagca ttctgttttg ttcaggatgg ctataatgat aaaatctttt caatagatat 2700 atagctaatt gtctcgt 2717 <210> SEQ ID NO 16 <211> LENGTH: 635 <212> TYPE: PRT <213> ORGANISM: Oryza sativa <400> SEQUENCE: 16 Met Pro Ala Arg Ser Ala Val Val Val Ile Ala Met Glu Pro Ser Ser 1 5 10 15 Ser Ile Thr Ile Ala Ser Ser Ser Ser Tyr Leu Ser Asn Gly Ser Ser 20 25 30 Pro Cys Ser Val Ser Leu Ala Pro Pro Gly Ala Gly Ala Val Ala Ala 35 40 45 Gln Ala Ala Pro Val Ala Ala Gly Glu Gly Gly Gly Gly Gly Gly Gly 50 55 60 Gly Gly Gly Gly Gly Ser Ser Ser Val Glu Val Val Ser Leu Asn Arg 65 70 75 80 Leu Ser Ala Asn Leu Glu Arg Leu Leu Leu Asp Ser Asp Leu Asp Cys 85 90 95 Ser Asp Ala Asp Val Asp Val Ala Asp Gly Gly Pro Pro Val Pro Val 100 105 110 His Arg Cys Ile Leu Ala Ala Arg Ser Thr Phe Phe Tyr Asn Leu Phe 115 120 125 Ala Ala Arg Gly Arg Gly Gly Asp Gly Ala Ala Gly Gly Gly Gly Gly 130 135 140 Gly Gly Gly Gly Gly Gly Glu Arg Thr Gly Gly Arg Pro Arg Tyr Lys 145 150 155 160 Met Glu Glu Leu Val Pro Gly Gly Arg Val Gly Arg Asp Ala Phe Leu 165 170 175 Ser Leu Leu Gly Tyr Leu Tyr Thr Gly Lys Leu Arg Pro Ala Pro Asp 180 185 190 Asp Val Val Ser Cys Ala Asp Pro Met Cys Pro His Asp Ser Cys Pro 195 200 205 Pro Ala Ile Arg Phe Asn Val Glu Gln Met Tyr Ala Ala Trp Ala Phe 210 215 220 Lys Ile Thr Glu Leu Ile Ser Leu Phe Gln Arg Arg Leu Leu Asn Phe 225 230 235 240 Val Asp Lys Thr Leu Val Glu Asp Val Leu Pro Ile Leu Gln Val Ala 245 250 255 Phe His Ser Glu Leu Thr Pro Val Leu Glu Lys Cys Ile Arg Arg Ile 260 265 270 Ala Arg Ser Asn Leu Asp Asn Val Ser Leu Asp Lys Glu Leu Pro Pro 275 280 285 Glu Val Ala Val Gln Ile Lys Glu Ile Arg Gln Lys Ser Gln Pro Asn 290 295 300 Glu Gly Asp Thr Val Ile Ser Asp Pro Val His Glu Lys Arg Val Arg 305 310 315 320 Arg Ile His Arg Ala Leu Asp Ser Asp Asp Val Glu Leu Val Lys Leu 325 330 335 Leu Leu Asn Glu Ser Glu Ile Thr Leu Asp Asp Ala Asn Ala Leu His 340 345 350 Tyr Ala Ala Ala Tyr Cys Asp Ser Lys Val Val Ser Glu Leu Leu Asp 355 360 365 Leu Arg Leu Ala Asn Leu Asn Leu Lys Asn Ser Arg Gly Tyr Thr Ala 370 375 380 Leu His Leu Ala Ala Met Arg Arg Glu Pro Ala Ile Ile Met Cys Leu 385 390 395 400 Leu Asn Lys Gly Ala Ala Val Ser Gln Leu Thr Ala Asp Gly Gln Ser 405 410 415 Ala Met Ser Ile Cys Arg Arg Leu Thr Arg Met Lys Asp Tyr Asn Thr 420 425 430 Lys Met Glu Gln Gly Gln Glu Ser Asn Lys Asp Arg Leu Cys Ile Asp 435 440 445 Ile Leu Asp Arg Glu Met Ile Arg Lys Pro Met Ala Val Glu Asp Ser 450 455 460 Val Thr Ser Pro Leu Leu Ala Asp Asp Leu His Met Lys Leu Leu Tyr 465 470 475 480 Leu Glu Asn Arg Val Ala Phe Ala Arg Leu Phe Phe Pro Ala Glu Ala 485 490 495 Lys Val Ala Met Gln Ile Ala Gln Ala Asp Thr Thr Pro Glu Phe Gly 500 505 510 Ile Val Pro Ala Ala Ser Thr Ser Gly Lys Leu Lys Glu Val Asp Leu 515 520 525 Asn Glu Thr Pro Val Thr Gln Asn Lys Arg Leu Arg Ser Arg Val Asp 530 535 540 Ala Leu Met Lys Thr Val Glu Leu Gly Arg Arg Tyr Phe Pro Asn Cys 545 550 555 560 Ser Gln Val Leu Asp Lys Phe Leu Glu Asp Asp Leu Pro Asp Ser Pro 565 570 575 Asp Ala Leu Asp Leu Gln Asn Gly Thr Ser Asp Glu Gln Asn Val Lys 580 585 590 Arg Met Arg Phe Cys Glu Leu Lys Glu Asp Val Arg Lys Ala Phe Ser 595 600 605 Lys Asp Arg Ala Asp Asn Ser Met Phe Ser Ile Leu Ser Ser Ser Ser 610 615 620 Ser Ser Ser Pro Pro Pro Lys Val Ala Lys Lys 625 630 635 <210> SEQ ID NO 17 <211> LENGTH: 593 <212> TYPE: PRT <213> ORGANISM: Arabidopsis thaliana <400> SEQUENCE: 17 Met Asp Thr Thr Ile Asp Gly Phe Ala Asp Ser Tyr Glu Ile Ser Ser 1 5 10 15 Thr Ser Phe Val Ala Thr Asp Asn Thr Asp Ser Ser Ile Val Tyr Leu 20 25 30 Ala Ala Glu Gln Val Leu Thr Gly Pro Asp Val Ser Ala Leu Gln Leu 35 40 45 Leu Ser Asn Ser Phe Glu Ser Val Phe Asp Ser Pro Asp Asp Phe Tyr 50 55 60 Ser Asp Ala Lys Leu Val Leu Ser Asp Gly Arg Glu Val Ser Phe His 65 70 75 80 Arg Cys Val Leu Ser Ala Arg Ser Ser Phe Phe Lys Ser Ala Leu Ala 85 90 95 Ala Ala Lys Lys Glu Lys Asp Ser Asn Asn Thr Ala Ala Val Lys Leu 100 105 110 Glu Leu Lys Glu Ile Ala Lys Asp Tyr Glu Val Gly Phe Asp Ser Val 115 120 125 Val Thr Val Leu Ala Tyr Val Tyr Ser Ser Arg Val Arg Pro Pro Pro 130 135 140 Lys Gly Val Ser Glu Cys Ala Asp Glu Asn Cys Cys His Val Ala Cys 145 150 155 160 Arg Pro Ala Val Asp Phe Met Leu Glu Val Leu Tyr Leu Ala Phe Ile 165 170 175 Phe Lys Ile Pro Glu Leu Ile Thr Leu Tyr Gln Arg His Leu Leu Asp 180 185 190 Val Val Asp Lys Val Val Ile Glu Asp Thr Leu Val Ile Leu Lys Leu 195 200 205 Ala Asn Ile Cys Gly Lys Ala Cys Met Lys Leu Leu Asp Arg Cys Lys 210 215 220 Glu Ile Ile Val Lys Ser Asn Val Asp Met Val Ser Leu Glu Lys Ser 225 230 235 240 Leu Pro Glu Glu Leu Val Lys Glu Ile Ile Asp Arg Arg Lys Glu Leu 245 250 255 Gly Leu Glu Val Pro Lys Val Lys Lys His Val Ser Asn Val His Lys 260 265 270 Ala Leu Asp Ser Asp Asp Ile Glu Leu Val Lys Leu Leu Leu Lys Glu 275 280 285 Asp His Thr Asn Leu Asp Asp Ala Cys Ala Leu His Phe Ala Val Ala 290 295 300 Tyr Cys Asn Val Lys Thr Ala Thr Asp Leu Leu Lys Leu Asp Leu Ala 305 310 315 320 Asp Val Asn His Arg Asn Pro Arg Gly Tyr Thr Val Leu His Val Ala 325 330 335 Ala Met Arg Lys Glu Pro Gln Leu Ile Leu Ser Leu Leu Glu Lys Gly 340 345 350 Ala Ser Ala Ser Glu Ala Thr Leu Glu Gly Arg Thr Ala Leu Met Ile 355 360 365 Ala Lys Gln Ala Thr Met Ala Val Glu Cys Asn Asn Ile Pro Glu Gln 370 375 380 Cys Lys His Ser Leu Lys Gly Arg Leu Cys Val Glu Ile Leu Glu Gln 385 390 395 400 Glu Asp Lys Arg Glu Gln Ile Pro Arg Asp Val Pro Pro Ser Phe Ala 405 410 415 Val Ala Ala Asp Glu Leu Lys Met Thr Leu Leu Asp Leu Glu Asn Arg 420 425 430 Val Ala Leu Ala Gln Arg Leu Phe Pro Thr Glu Ala Gln Ala Ala Met 435 440 445 Glu Ile Ala Glu Met Lys Gly Thr Cys Glu Phe Ile Val Thr Ser Leu 450 455 460 Glu Pro Asp Arg Leu Thr Gly Thr Lys Arg Thr Ser Pro Gly Val Lys 465 470 475 480 Ile Ala Pro Phe Arg Ile Leu Glu Glu His Gln Ser Arg Leu Lys Ala 485 490 495 Leu Ser Lys Thr Val Glu Leu Gly Lys Arg Phe Phe Pro Arg Cys Ser 500 505 510 Ala Val Leu Asp Gln Ile Met Asn Cys Glu Asp Leu Thr Gln Leu Ala 515 520 525 Cys Gly Glu Asp Asp Thr Ala Glu Lys Arg Leu Gln Lys Lys Gln Arg 530 535 540 Tyr Met Glu Ile Gln Glu Thr Leu Lys Lys Ala Phe Ser Glu Asp Asn 545 550 555 560 Leu Glu Leu Gly Asn Ser Ser Leu Thr Asp Ser Thr Ser Ser Thr Ser 565 570 575 Lys Ser Thr Gly Gly Lys Arg Ser Asn Arg Lys Leu Ser His Arg Arg 580 585 590 Arg 

What is claimed is:
 1. An isolated polynucleotide that encodes an NPR1 polypeptide having a sequence identity of at least 80% based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and
 16. 2. The polynucleotide of claim 1 wherein the sequence identity is at least 85%.
 3. The polynucleotide of claim 1 wherein the sequence identity is at least 90%.
 4. The polynucleotide of claim 1 wherein the sequence identity is at least 95%.
 5. The polynucleotide of claim 1 wherein the polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and
 16. 6. The polynucleotide of claim 1 wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, and
 15. 7. The polynucleotide of claim 1 wherein the polypeptide is an NPR1.
 8. An isolated complement of the polynucleotide of claim 1, wherein (a) the complement and the polynucleotide consist of the same number of nucleotides, and (b) the nucleotide sequences of the complement and the polynucleotide have 100% complementarity.
 9. An isolated nucleic acid molecule that (1) comprises at least 100 nucleotides and (2) remains hybridized with the isolated polynucleotide of claim 1 under a wash condition of 0.1×SSC, 0.1% SDS, and 65° C.
 10. A cell comprising the polynucleotide of claim
 1. 11. The cell of claim 10, wherein the cell is selected from the group consisting of a yeast cell, a bacterial cell and a plant cell.
 12. A virus comprising the polynucleotide of claim
 1. 13. A transgenic plant comprising the polynucleotide of claim
 1. 14. A method for transforming a cell, comprising introducing into a cell the polynucleotide of claim
 1. 15. A method for producing a transgenic plant comprising (a) transforming a plant cell with the polynucleotide of claim 1, and (b) regenerating a plant from the transformed plant cell.
 16. A method for producing a polynucleotide fragment comprising (a) selecting a nucleotide sequence comprised by the polynucleotide of claim 1, and (b) synthesizing a polynucleotide fragment containing the nucleotide sequence.
 17. The method of claim 16, wherein the fragment is produced in vivo.
 18. An isolated NPR1 polypeptide that has a sequence identity of at least 80% based on the Clustal method compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and
 16. 19. The isolated polypeptide of claim 18 wherein the sequence identity is at least 85%.
 20. The isolated polypeptide of claim 18 wherein the sequence identity is at least 90%.
 21. The isolated polypeptide of claim 18 wherein the sequence identity is at least 95%.
 22. The polypeptide of claim 18 wherein the polypeptide has a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and
 16. 23. The polypeptide of claim 18, wherein the polypeptide is aNPR1 .
 24. A chimeric gene comprising the polynucleotide of claim 1 operably linked to at least one regulatory sequence.
 25. A method for altering the level of pathogen resistance in a plant, the method comprising the steps of: (a) transforming a plant cell with a chimeric gene containing the polypeptide of claim 1; (b) culturing the transformed plant cell under conditions suitable for the expression of the chimeric gene; (c) maintaining the plant cell under conditions that are suitable for its development into a plant; and (d) comparing the level of pathogen resistance of the plant cell containing the polynucleotide of claim 1 and a plant cell not containing the polynucleotide of claim
 1. 